Method of treating acute coronary syndromes

ABSTRACT

The present invention relates to methods and compositions designed for the treatment or management of acute coronary syndromes, particularly, unstable angina and acute myocardial infarction. The methods of the invention comprise the administration of an effective amount of a formulation containing one or more therapeutic agents which specifically decreases or inhibits the activity of phagocytic cells and/or eliminates or diminishes the amount of phagocytic cells including, but not limited to, macrophages and monocytes. The formulations are specifically targeted to phagocytic cells. The invention also provides pharmaceutical compositions of formulations containing one or more therapeutic agents of the invention for administration to subjects currently suffering from or having recently suffered an acute coronary syndrome such as unstable angina and acute myocardial infarction.

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 10/607,623 filed Jun. 27, 2003, which is incorporated byreference herein in its entirety.

1. FIELD OF INVENTION

[0002] The present invention relates to methods and compositionsdesigned for the treatment or management of acute coronary syndromes,particularly, unstable angina and acute myocardial infarction. Themethods of the invention comprise the administration of an effectiveamount of a formulation containing one or more therapeutic agents whichspecifically decreases or inhibits the activity of and/or eliminates ordiminishes the amount of phagocytic cells including, but not limited to,macrophages and monocytes.

2. BACKGROUND OF THE INVENTION

[0003] Coronary artery disease is a leading cause of death inindustrialized countries. In the United States, 50-60% of heart attacksoccur in people without documented coronary artery disease. A chiefcontributor to the pathology of the disease is the formation ofatherosclerotic plaques. Atherosclerotic plaques are thickened areas invessel walls which result from an accumulation of cholesterol,proliferating smooth muscle cells, and inflammatory cells.

[0004] Atherosclerotic Plaques

[0005] In general, an atherosclerotic plaque consists of a raised focalpoint within the intima having a central core of extra-cellular lipidscovered by a fibrous cap. The core within the plaque containscrystalline cholesterol, cholesterol esters, phospholipids, cellulardegradation products and collagen remnants. The fibrous cap separatesthe core of the plaque from the lumen of the blood vessel or artery andis comprised mainly of connective tissues that are a dense, fibrousextracellular matrix made up of collagens, elastins, proteoglycans andother extracellular matrix materials. The fibrous cap varies inthickness, number of smooth muscle cells and macrophages, and collagencontent. (Vallabhajosula et al., 1997, J. Nucl. Med. 38(11): 1788-1796).

[0006] Atherosclerotic plaques can be characterized as active and proneto rupture (“vulnerable or high-risk plaques”) or inactive andrelatively stable (“stable plaques”). A vulnerable, high-risk orrupture-prone plaque is characterized by an abundance of inflammatorycells (such as macrophages), a thin fibrous cap, and a large lipid core.The size of the lipid pool within the atherosclerotic plaque and thethickness of the overlying fibrous cap are important characteristicspredicting the stability of the plaque. The edge of the fibrous cap (theshoulder region) is a location of high stress and predisposed torupture, in part, due to the accumulation of inflammatory cells (such asmacrophages) in the area and their secretion of enzymes that causedegradation of the material that makes up the fibrous cap (Moreno et al,Circulation., 1994, 90:775-8; van der Wal et al., 1994, Circulation89:36-44.; Jander et al, 1998, Stroke, 29:1625-1630) which can lead torupture of the plaque.

[0007] Rupture of the lipid-laden plaque exposes the highly thrombogeniccore and the sub-endothelial vascular smooth muscle cell component ofthe arterial wall to circulating blood. Platelet activation, adhesionand aggregation follow this almost immediately. Platelet adhesion andactivation results in the release of coagulation factors and theinitiation of the coagulation cascade. The released growth factors,specifically platelet-derived growth factor (PDGF) stimulate theproliferation and migration of vascular smooth muscle cells.Proliferation and migration of vascular smooth muscle cells can lead toplaque remodeling and increased vascular stenosis, or interact with theplatelets leading to enhanced thrombogenesis (Pasterkamp et al., 2000,J. Clin. Basic Cardiol. 3:81-86). The resulting thrombosis caused by thevulnerable plaque can cause unstable angina, acute myocardialinfarction, stroke, acute deterioration in peripheral artery disease, orsudden coronary death.

[0008] Unstable Angina

[0009] The heart requires oxygen-rich blood to function. The right andleft coronary arteries branch from the aorta and carry oxygenated bloodto the tissues of the heart. When the coronary arteries fail to deliveran adequate amount of oxygen-rich blood (a condition called hypoxia) tothe heart, chest pain, pressure, or discomfort, commonly known asangina, result. If this situation is prolonged, oxygen depravation candamage the heart muscle itself (a situation known as ischemia) eitherreversibly or irreversibly.

[0010] Angina is classified broadly as stable or unstable, depending onits severity and pattern of occurrence. Stable angina occurs whenincreased physical activity (e.g., hurrying across a street or climbinga long flight of stairs) raises the demand for oxygen-rich blood. Due toa possible multitude of factors (the most common of which is one or moreoccluded coronary arteries), the supply created by the coronary bloodflow cannot meet this increased demand and hypoxia results. Unstableangina is understood as anginal pain that occurs with lesser degrees ofexertion, increasing frequency, or at rest (i.e., without exertion).Unstable angina that occurs at rest represents the condition in its mostserious form. It usually is caused by the formation of a blood clot in acoronary artery at the site of a ruptured plaque and, if left untreated,it may result in a heart attack and irreversible damage to the heart.

[0011] Unstable angina is likely due to the partial rupture of avulnerable plaque that has become unstable. The plaque's partial rupturecauses a thrombus to develop, but does not completely occlude theartery. Endogenous clot-fighting mechanisms serve to break up the clotbut, over time, the plaque continues to rupture and the clottingepisodes repeat. Although this patient may not have yet suffered amyocardial infarction, he or she is at high risk of doing so (e.g., ifthe unstable plaque completely ruptures or if the endogenous clotfighting mechanisms cannot eliminate the clot before total occlusion ofthe artery). Disrupted fibrous caps taken post mortem from patients withunstable angina are often more heavily infiltrated with macrophages atthe plaque rupture site than plaque from cases of stable angina.

[0012] Acute Myocardial Infarction

[0013] Acute myocardial infarction (“AMI”) refers to a common clinicalcondition that leads to necrosis of myocardial tissue. This condition iswell known in the art and is characterized by the occurrence of pain (inmost cases precordial), characteristic electrocardiographic changes, andan increase in plasma levels of intracellular enzymes (such ascreatinine phosphokinase and α-hydroxybutyrate dehydrogenase) or cardiacproteins (such as components of the troponin complex, and myoglobin)released by the necrotic cardiac tissue. AMI may be accompanied byhypotension, circulatory failure, pulmonary edema and arrhythmia. Inmost cases, but not exclusively, AMI results from vascular injury andthrombosis in the coronary vessels, which causes these vessels to becomeoccluded with subsequent impaired blood flow to the jeopardizedmyocardium (Fuster et al., 1992, New Engl. J. Med., 326:242-310). Inmostcases, the time of the occlusion of the coronary vessel can be estimatedfrom the medical history, the course of plasma levels of intracellularheart muscle enzymes and electrocardiographic changes.

[0014] The initiating event of many myocardial infarctions (heartattacks) is rupture of an atherosclerotic plaque. Such rupture mayresult in formation of a thrombus or blood clot in the coronary arterywhich supplies the infarct zone. The infarct zone or area, as it iscommonly referred to, is an area of necrosis which results from anobstruction of blood circulation. The thrombus formed is composed of acombination of fibrin and blood cells. The location, degree and durationof the occlusion caused by the clot determine the mass of the infarctzone and the extent of damage. Ultimately, the extent of myocardialdamage caused by the coronary occlusion depends upon the “territory”supplied by the affected vessel, the degree of occlusion of the vessel,the amount of blood supplied by collateral vessels to the affectedtissue, and the demand for oxygen of the myocardium whose blood supplyhas suddenly been limited (Pasternak and Braunwald, 1994, AcuteMyocardial Infarction, Harrison's Principles of Internal Medicine,13^(th) Ed., pgs. 1066-77).

[0015] Macrophages and the Inflammatory Response

[0016] Macrophages are involved in the cause and/or pathology of somecoronary syndromes. Macrophage secretion of proteolytic proteins thatdegrade the fibrous caps of plaques decrease cap thickness as well asincrease additional macrophage infiltration thus contributing to plaqueinstability. Therefore macrophages are considered to have a central rolein plaque rupture and their presence in large concentrations isconsidered predictive to such rupture. Indeed, erosion and/or disruptionof the fibrous cap of atherosclerotic plaques is known to modulatearterial thrombus formation, leading to the onset of acute ischemicevents. It is clear that rupture at the site of a vulnerableatherosclerotic plaque is the most frequent cause of acute coronarysyndromes, such as unstable angina, myocardial infarction or suddendeath.

[0017] Inflammation has been related both to the pathogenesis of acutemyocardial infarctions and to the healing and repair following AMI.Myocardial ischemia prompts an inflammatory response. In addition,reperfusion, the mainstay of current acute therapy of AMI, also enhancesinflammation. Reperfusion involves the rapid dissolution of theoccluding thrombus and the restoration of blood flow to the area of theheart which has had its blood supply cut off. The presence ofinflammatory cells in the ischemic myocardial tissues has traditionallybeen believed to represent the pathophysiological response to injury.However, experimental studies have shown that while crucial to healing,the influx of inflammatory cells into tissues, specifically macrophageswhich are phagocytic cells, results in tissue injury beyond that causedby ischemia alone.

[0018] Macrophages and other leukocytes infiltrate the myocardium soonafter ischemia ensues. Macrophages secrete several cytokines, whichstimulate fibroblast proliferation. However, the activated macrophagesalso secrete cytokines and other mediators that promote myocardialdamage. Accordingly, the influx of macrophages into the myocardiumincreases myocardial necrosis and expands the zone of infarct. Thus,although the acute phase of inflammation is a necessary response for thehealing process, persistent activation is in fact harmful to the infarctarea as well as the area surrounding it, the so-called ‘peri-infarctzone’.

[0019] The inflammatory response that follows myocardial ischemia iscritical in determining the severity of the resultant damage caused bythe activated macrophages. Plasma levels of inflammatory chemotacticfactors (macrophage chemoattractant protein-1 (MCP-1), macrophageinflammatory protein-1 alpha (MIP-1 alpha), have been shown to correlatewith subsequent heart failure and left ventricular dysfunction (see, forexample, Parissis, et al., 2002, J. Interferon Cytokine Res.,22(2):223-9). Peripheral monocytosis (an elevated number of monocytes)at two and three days after AMI is associated with left ventriculardysfunction and left ventricular aneurysm, suggesting a possible role ofmonocytes in the development of left ventricular remodeling afterreperfused AMI (Maekawa, Y. et al., 2002, J. Am. Coll. Cardiol.,39(2):241-6). Left ventricular remodeling after acute myocardialinfarction is the process of infarct expansions followed by progressiveleft ventricular dilation and is associated with an adverse clinicaloutcome. Furthermore, plasma levels of macrophage chemoattractantprotein-1(MCP-1) are elevated in patients with acute myocardialinfarction. MCP-1 is induced by myocardial ischemia/reperfusion injuryand neutralization of this chemokine significantly reduced infarct size.

[0020] Suppression of the inflammatory response by nonspecificanti-inflammatory composites after coronary occlusion, in severalcoronary occlusion/reperfusion models, was shown to reduce the infarctarea (See, for example, Squadrito, et al., 1997, Eur. J. Pharmacol.;335:185-92; Libby, et al., 1973, J. Clin. Invest., 3:599-607; Spath, etal., 1974, Circ. Res., 35: 44-51). However, these nonspecific regimensare associated with adverse effects, such as interference with scarformation and healing, and, in some patients, the development ofaneurysm and rupture of the ventricular wall. As such, these regimensare precluded from clinical use. However, animal models that have adecreased ability to suppress macrophage function due to a deficiency inthe anti-inflammatory cytokine interleukin-10 were shown to suffer fromincreased infarct size and myocardial necrosis in a coronary occlusionmodel (Yang, Z. et al., 2000, Circulation, 101:1019-1026.)

[0021] One object of the present invention is the identification oftherapeutic agents capable of blocking the accumulation of and/or thebiological function including secretion of factors from phagocytic cells(particularly macrophages and monocytes) in the patient suffering froman acute coronary syndrome (particularly unstable angina or and acutemyocardial infarction).

[0022] Another object of the invention is the development of methods fortreating an acute coronary syndrome (particularly unstable angina or andacute myocardial infarction) as well as stabilizing the plaquesassociated with these syndromes.

3. SUMMARY OF THE INVENTION

[0023] The present invention relates to methods and compositionsdesigned for the treatment or management of acute coronary syndromes,particularly, unstable angina and acute myocardial infarction. Themethods of the invention comprise the administration of an effectiveamount of a formulation containing one or more therapeutic agents whichspecifically inhibits the activity of and/or diminishes the amount ofphagocytic cells including, but not limited to, macrophages andmonocytes. Administration of a formulation containing one or moretherapeutic agents according to the invention acts as an acute,treatment aimed at stabilizing the patient's coronary syndromecondition. In one embodiment, a formulation containing one or moretherapeutic agents is administered to a patient suffering from unstableangina to stabilize a vulnerable or unstable plaque. In anotherembodiment, a formulation is administered to a patient currentlysuffering or recently having suffered an acute myocardial infarction tominimize infarct size and myocardial necrosis.

[0024] In preferred embodiments, the formulation specifically targetsphagocytic cells. Because phagocytic cells possess the unique ability ofphagocytosis, in these embodiments, the formulations are prepared suchthat they comprise particles of such properties as to enter into a cellprimarily or exclusively via phagocytosis. The formulation may comprisean encapsulated therapeutic agent, an embedded therapeutic agent, or aparticulate therapeutic agent. Once phagocytosed, the therapeutic agentis released from the formulation into the targeted phagocytic cells,e.g., macrophages and monocytes, and inhibits the function of and/ordestroys the phagocytic cell.

[0025] In one embodiment, the present invention relates to a method oftreating an acute coronary syndrome by administering to an individual inneed thereof an effective amount of a formulation comprising anencapsulated therapeutic agent. The therapeutic agent is encapsulated ina suitable carrier of a specific dimension. The formulation specificallytargets phagocytic cells by virtue of its properties, such as, forexample, size or charge, which allow the formulation to be taken-upprimarily or exclusively by phagocytosis. Once the formulation istaken-up by the phagocytic cell, the encapsulated therapeutic agent isreleased and the agent is able to inhibit the activity of and/or destroythe phagocytic cell.

[0026] In another embodiment, the present invention relates to a methodof treating an acute coronary syndrome by administering to an individualin need thereof an effective amount of a formulation comprising anembedded therapeutic agent. The therapeutic agent is embedded in asuitable carrier of a specific dimension. The formulation specificallytargets phagocytic cells by virtue of its properties, such as, forexample, size and/or charge, which allow the formulation to be taken-upprimarily or exclusively by phagocytosis. Once inside the phagocyticcells the embedded therapeutic agent is released and the agent is ableto inhibit the activity of and/or destroy the phagocytic cell.

[0027] In another embodiment, the present invention relates to a methodof treating an acute coronary syndrome by administering to an individualin need thereof an effective amount of a formulation comprising aparticulate therapeutic agent. The therapeutic agent is made intoparticulates of a specific dimension. The formulation specificallytargets phagocytic cells by virtue of the particulate's properties, suchas, for example, size and/or charge, which allow the formulation to betaken-up primarily or exclusively by phagocytosis. Once inside thephagocytic cells the particulate therapeutic agent is able to inhibitthe activity of and/or destroy the phagocytic cell.

[0028] The present invention also relates to a method of stabilizingplaques associated with an acute coronary syndrome by administering toan individual in need thereof an effective amount of a formulationcomprising an encapsulated, embedded, or particulate therapeutic agent.

[0029] In a further embodiment, the present invention includes apharmaceutical composition for administration to subjects currentlysuffering from or having recently suffered an acute coronary syndromesuch as unstable angina and acute myocardial infarction comprising aformulation selected from the group consisting of an encapsulatedtherapeutic agent, an embedded therapeutic agent, and a particulatetherapeutic agent together with a pharmaceutically acceptable vehicle,carrier, stabilizer or diluent for the treatment of an acute coronarysyndrome.

[0030] The formulation of present invention is preferably in the sizerange of 0.03-1.0 microns. However, depending on the type of agentand/or the carrier used, the more preferred ranges include, but are notlimited to, 0.07-0.5 microns, 0.1-0.3 microns and 0.1 to 0.18.

4. BRIEF DESCRIPTION OF THE FIGURES

[0031]FIG. 1 illustrates the effect of liposomal alendronate treatmenton the size of infarct area after transient coronary artery occlusion inrabbits. The size of the infarct zone was calculated as the area of theinfarcted zone as a % of the left ventricular area supplied by theoccluded artery and thus at risk for subsequent infarction. Data areexpressed as mean±SD, with n=4/group and a p value of p<0.05.

[0032]FIGS. 2A-2B illustrate the effect of liposomal alendronatetreatment on myocardial morphology after reversible coronary occlusionin rabbits. Control rabbits (A) have distorted myocardial morphologywhile rabbits treated with liposomal alendronate (B) have a more normalmyocardial morphology.

[0033]FIGS. 3A-3B illustrate the reduction in macrophage infiltrationfollowing treatment with liposomal alendronate after reversible coronaryocclusion in rabbits. Control rabbits (A) show increasedRAM11+macrophage accumulation in the zone of infarct as compared torabbits treated with liposomal alendronate (B).

5. DETAILED DESCRIPTION OF THE INVENTION

[0034] Phagocytic cells, particularly macrophages and monocytes, areinvolved in the cause and/or pathology of some coronary syndromes.Macrophages/monocytes degrade the fibrous caps of plaques through thesecretion of various substances that not only decrease cap thickness,but also serve to recruit additional macrophages/monocytes to the area.Degradation of the fibrous cap leads to exposure to blood of the lipidcore of the plaque as well as initiation of the clotting cascade whichculminates in a thrombus. The thrombus may partially occlude the lumenleading to unstable angina or it may completely occlude the lumen thuscausing an acute mycardial infarction. Once an acute myocardialinfarction occurs, macrophages/monocytes are recruited to the damagedmyocardial tissue and secrete cytokines and other mediators that promotemyocardial damage thus resulting in tissue injury beyond that caused byischemia alone and increases myocardial necrosis which expands the zoneof infarct. Although a complete and chronic incapacitation and/orablation of phagocytic cells is not desirable, such a decrease inphagocytic cell activity and/or presence is desirable in the short termduring or after an acute coronary syndrome to stabilize the patientand/or reduce the damage of the coronary syndrome.

[0035] The present invention relates to methods and compositionsdesigned to decrease or inhibit the activity of and/or eliminate ordiminish the amount of phagocytic cells (including, but not limited to,macrophages and monocytes) for an acute, short term period during orfollowing an acute coronary syndrome for the treatment or management ofthe acute coronary syndrome (including, but not limited to, unstableangina and acute myocardial infarction). The methods of the inventioncomprise the administration of an effective amount of a formulationcontaining one or more therapeutic agents which specifically decreasesor inhibits the activity of and/or eliminates or diminishes the amountof phagocytic cells (including, but not limited to, macrophages andmonocytes) in a patient. Administration of a formulation containing oneor more therapeutic agents is contemplated as an acute, short termtreatment aimed at stabilization of the patient and/or minimization ofthe immediate and long term damage from the acute coronary syndrome. Inone embodiment, a formulation containing one or more therapeutic agentsare administered to a patient suffering from unstable angina tostabilize a vulnerable or unstable plaque and decrease the immediatethreat of an acute myocardial infarction. In another embodiment, one ormore therapeutic agents are administered to a patient currentlysuffering or recently having suffered an acute myocardial infarction tominimize the infarct size and myocardial necrosis.

[0036] The formulations used in the methods of the inventionspecifically decrease or inhibit the activity of phagocytic cells and/oreliminate or diminish the amount of phagocytic cells in a patient.Specificity of the formulation is due to the ability of the compositionto affect only particular cell types (e.g., macrophages and/ormonocytes). In preferred embodiments, specificity of the formulation forphagocytic cells is due to the physiochemical properties, e.g. size orcharge, of the formulation such that it can only or primarily beinternalized by phagocytosis. Once phagocytosed and intracellular, thetherapeutic agent inhibits or decreases the activity of the phagocyticcell and/or destroys the phagocytic cell. Although not intending to bebound by any particular mechanism of action, the therapeutic agents ofthe formulation are released upon becoming intracellular beforedisabling an/or destroying the phagocytic cell.

[0037] The formulation of the present invention, e.g., the encapsulatedtherapeutic agent, embedded therapeutic agent or the particulatetherapeutic agent, suppresses the inflammatory response by transientlydepleting and/or inactivating cells that are important triggers in theinflammatory response, namely macrophages and/or monocytes. Theencapsulated agent, embedded agent and/or particulate agent aretaken-up, by way of phagocytosis, by the macrophages and monocytes. Incontrast, non-phagocytic cells are incapable of taking up theformulation due to the large dimension and/or other physiochemicalproperties of the formulation.

[0038] The term “phagocytosis” as used herein refers to a preferredmeans of entry into a phagocytic cell and is well understood in the art.However, the term should be understood to also encompass other forms ofendocytosis which may also accomplish the same effect. In particular, itis understood that pinocytosis, receptor-mediated endocytosis and othercellular means for absorbing/internalizing material from outside thecell are also encompassed by the methods and compositions of the presentinvention.

[0039] The invention also provides pharmaceutical compositionscomprising one or more therapeutic agents of the invention foradministration to subjects currently suffering from or recently havingsuffered an acute coronary syndrome such as unstable angina and acutemyocardial infarction.

5.1 Therapeutic Agents

[0040] The therapeutic agents used in the formulations and in themethods of the invention specifically decrease or inhibit the activityof phagocytic cells and/or eliminate or diminish the amount ofphagocytic cells in a patient, by virtue of the physiochemicalproperties, such as size or charge, of the formulation. The therapeuticagent may be an intra-cellular inhibitor, deactivator, toxin, arrestingsubstance and/or cytostatic/cytotoxic substance that, once inside aphagocytic cell such as a macrophage or monocyte, inhibits, destroys,arrests, modifies and/or alters the phagocytic cell such that it can nolonger function normally and/or survive.

[0041] As used herein, the term “therapeutic agents” refers to moleculeswhich either make up the formulation or form a part of the formulationand provide the inactivating/toxic potency to the formulation., e.g.,inhibits or decreases phagocytic cell activity and/or eliminates ordecreases the amount of phagocytic cells. Compounds that can betherapeutic agents include, but are not limited to, inorganic or organiccompounds; or a small molecule (less than 500 daltons) or a largemolecule, including, but not limited to, inorganic or organic compounds;proteinaceous molecules, including, but not limited to, peptide,polypeptide, protein, post-translationally modified protein, antibodiesetc.; or a nucleic acid molecule, including, but not limited to,double-stranded DNA, single-stranded DNA, double-stranded RNA,single-stranded RNA, or triple helix nucleic acid molecules. Compoundscan be natural products derived from any known organism (including, butnot limited to, animals, plants, bacteria, fungi, protista, or viruses)or from a library of synthetic molecules. Therapeutic agents can bemonomeric as well as polymeric compounds.

[0042] In preferred embodiments where the preferred therapeutic agentmay be a bisphosphonate or analog thereof. The term “bisphosphonate” asused herein, denotes both geminal and non-geminal bisphosphonates. In aspecific embodiment, the bisphosphonate has the following formula (I):

[0043] wherein R₁ is H, OH or a halogen atom; and R₂ is halogen; linearor branched C₁-C₁₀ alkyl or C₂-C₁₀ alkenyl optionally substituted byheteroaryl or heterocyclyl C₁-C₁₀ alkylamino or C₃-C₈ cycloalkylaminowhere the amino may be a primary, secondary or tertiary; —NHY where Y ishydrogen, C₃-C₈ cycloalkyl, aryl or heteroaryl; or R₂ is —SZ where Z ischlorosubstituted phenyl or pyridinyl.

[0044] In a more specific embodiment, the bisphosphoate is alendronateor an analog thereof. In such an embodiment, the alendronate has thefollowing formula (II):

[0045] In other specific embodiments, additional bisphosphonates can beused in the methods of the invention. Examples of other bisphosphonatesinclude, but are not limited to, clodronate, tiludronate,3-(N,N-dimethylamino)-1-hydroxypropane-1,1-diphosphonic acid, e.g.dimethyl-APD; 1-hydroxy-ethylidene-1,1-bisphosphonic acid, e.g.etidronate; 1-hydroxy-3(methylpentylamino)-propylidene-bisphosphonicacid, (ibandronic acid), e.g. ibandronate;6-amino-1-hydroxyhexane-1,1-diphosphonic acid, e.g. amino-hexyl-BP;3-(N-methyl-N-pentylamino)-1-hydroxypropane-1,1-diphosphonic acid, e.g.methyl-pentyl-APD; 1-hydroxy-2-(imidazol-1-yl)ethane-1,1-diphosphonicacid, e.g. zoledronic acid;1-hydroxy-2-(3-pyridyl)ethane-1,1-diphosphonic acid (risedronic acid),e.g. risedronate;3-[N-(2-phenylthioethyl)-N-methylamino]-1-hydroxypropane-1,1-bishosphonicacid; 1-hydroxy-3-(pyrrolidin-1-yl)propane-1,1-bisphosphonic acid,1-(N-phenylaminothiocarbonyl)methane-1,1-diphosphonic acid, e.g. FR78844 (Fujisawa); 5-benzoyl-3,4-dihydro-2H-pyrazole-3,3-diphosphonicacid tetraethyl ester, e.g. U81581 (Upjohn); and1-hydroxy-2-(imidazo[1,2-a]pyridin-3-yl)ethane-1,1-diphosphonic acid,e.g. YM 529, or analogs thereof.

[0046] Other formulations containing therapeutic agents include, but arenot limited to, gallium, gold, selenium, gadolinium, silica,mithramycin, paclitaxel, sirolimus, everolimus, and other similaranalogs thereof. Generally, chemotherapeutic agents, such as, forexample, 5-fluorouracil, cisplatinum, alkylating agents and otheranti-proliferation or anti-inflammatory compounds, such as, for example,steroids, aspirin and non-steroidal anti-inflammatory drugs may also beused in a formulation.

[0047] The present invention is meant to encompass the administration ofone or more formulations to manage or treat an acute coronary syndrome.More than one formulation can be administered in combination to thepatient. The term “in combination” is not limited to the administrationof the formulation at exactly the same time, but rather it is meant thatthe formulations are administered to a patient in a sequence and withina time interval such that they can act together to provide an increasedbenefit than if they were administered otherwise. For example, eachformulation may be administered at the same time or sequentially in anyorder at different points in time; however, if not administered at thesame time, they should be administered sufficiently close in time so asto provide the desired therapeutic effect. Each formulation can beadministered separately, in any appropriate form and by any suitableroute which effectively transports the therapeutic agent to theappropriate or desirable site of action. Preferred modes ofadministration include intravenous (IV) and intra-arterial (IA). Othersuitable modes of administration include intramuscular (IM),subcutaneous (SC), and intraperitonal (IP) and oral (PO). Suchadministration may be bolus injections or infusions. Another mode ofadministration may be by perivascular delivery. The formulation may beadministered directly or after dilution. Combinations of any of theabove routes of administration may also be used in accordance with theinvention.

[0048] In various embodiments, the formulations are administered lessthan 1 hour apart, at about 1 hour apart, at about 1 hour to about 2hours apart, at about 2 hours to about 3 hours apart, at about 3 hoursto about 4 hours apart, at about 4 hours to about 5 hours apart, atabout 5 hours to about 6 hours apart, at a about 6 hours to about 7hours apart, at about 7 hours to about 8 hours apart, at about 8 hoursto about 9 hours apart, at about 9 hours to about 10 hours apart, atabout 10 hours to about 11 hours apart, at about 11 hours to about 12hours apart, no more than 24 hours apart or no more than 48 hours apart.In one embodiment two or more formulations are administered concurrentlyor within the same patient visit.

5.1.1 Identification of Therapeutic Agents

[0049] The invention provides methods of screening for compounds thatcan be used as a therapeutic agent. Although not intending to be boundby a particular mechanism of action, a compound that is a therapeuticagent for use in the methods of the invention can, once targeted to thephagocytic cell by the physiochemical properties of the formulationitself, i) inhibit phagocytic cell activity, ii) decrease phagocyticcell activity, iii) eliminate phagocytic cells from circulation and/orfrom the area affected by the acute coronary syndrome, and/or iv)decrease the number of phagocytic cells in circulation and/or in thearea affected by the acute coronary syndrome.

[0050] The methods of screening for therapeutic agents generally involveincubating a candidate compound with phagocytic cells either in vitro orin vivo and then assaying for an alteration (e.g., decrease) inphagocytic cell activity or longevity thereby identifying a compoundthat is a therapeutic agent for use in the present invention. Any methodknown in the art can be used to assay phagocytic cell activity orlongevity. In one embodiment, phagocytic cell activity is assayed by thelevel of cell activation in response to an activating stimulus. Forexample, macrophage/monocyte activation can be assayed by quantifyingthe levels of chemotactic factors such as macrophage chemoattractantprotein-1 (MCP-1) and macrophage inflammatory protein-1 alpha (MIP-1alpha) as well as other substances produced by macrophages such asinterleukin 1 beta (IL-1β) and tissue necrosis factor alpha (TNF-αx). Inanother embodiment, phagocytic cell longevity is assayed. For example,cell proliferation can be assayed by measuring ³H-thymidineincorporation, by direct cell count, by detecting changes intranscriptional activity of known genes such as proto-oncogenes (e.g.,fos, myc) or cell cycle markers; or by trypan blue staining. Any methodknown in the art can be used to assay for levels of mRNA transcripts(e.g., by northern blots, RT-PCR, Q-PCR, etc.) or protein levels (e.g.,ELISA, western blots, etc.).

[0051] In one embodiment, a compound that decreases the activity of aphagocytic cell is identified by:

[0052] a) contacting a phagocytic cell with a first compound and asecond compound, said first compound being a compound which activatessaid phagocytic cell and said second compound being a candidatecompound; and

[0053] b) determining the level of activation in said contactedphagocytic cell, wherein a decrease in activation in said contacted cellas compared to the level of activation in a phagocytic cell contactedwith said first compound in the absence of said second (i.e., a controlcell) indicates that said second compound decreases the activity of aphagocytic cell.

[0054] In another embodiment, a compound that decreases the amount ofphagocytic cells is identified by:

[0055] a) contacting a phagocytic cell with a compound; and

[0056] b) determining the viability of said contacted phagocytic cell,wherein a decrease in viability in said contacted cell as compared tothe viability of a phagocytic cell not contacted with said compound(i.e., a control cell) indicates that said compound decreases the amountof phagocytic cells.

[0057] In other embodiments, candidate compounds are assayed for theirability to alter phagocytic cell activity or longevity in a manner thatis substantially similar to or better than compounds known to alterphagocytic cell activity or longevity in a therapeutically desirableway. As used herein “substantially similar to” refers to an agent havingsimilar action on a phagocytic cell as an exemplified agent, i.e., anagent that inhibits the activity, function, motility, and/or depletionof phagocytic cells.

[0058] Additionally, candidate compounds can be used in animal models ofacute coronary syndromes to assess their ability to be used in themethods of the invention. In one embodiment, a rabbit AMI model can beused (see e.g., Section 6.1).

5.2 Formulation of Therapeutic Agents

[0059] Formulations containing one or more therapeutic agents can beprepared so that the size of the formulation is large enough to only orprimarily be internalized by phagocytosis, thus imparting specificity tophagocytic cells. Although non-phagocytic cells may be affected by sucha formulation should it become intracellular, there is no mechanism fora non-phagocytic cell to internalize a formulation prepared in thismanner. Formulations imparting extrinsic specificity to one or moretherapeutic agents are preferably in the size range of 0.03-1.0 microns,more preferably 0.07-0.5 microns, more preferably 0.1-0.3 microns, andmore preferably 0.1 to 0.18 microns.

[0060] Any method known in the art can be used to incorporate atherapeutic agent into a formulation such that it can only or primarilybe internalized via phagocytosis. Formulations of therapeutic agents maysequester the therapeutic agents for a sufficient time to enhancedelivery of the agent to the target site. Furthermore, formulations oftherapeutic agents may discharge the therapeutic agent from theparticles when they are within the target cell (e.g., the phagocyticcell) at the target site.

[0061] In one embodiment, the therapeutic agent is encapsulated in acarrier (i.e., encapsulating agent) of desired properties. In a specificembodiment, the encapsulating agent is a liposome. The liposomes may beprepared by any of the methods known in the art (see, e.g., Mönkkönen,J. et al., 1994, J. Drug Target, 2:299-308; Mönkkönen, J. et al., 1993,Calcif. Tissue Int., 53:139-145; Lasic D D., Liposomes Technology Inc.,Elsevier, 1993, 63-105. (chapter 3); Winterhalter M, Lasic D D, ChemPhys Lipids, 1993 September;64(1-3):35-43). The liposomes may bepositively charged, neutral or, more preferably, negatively charged. Theliposomes may be a single lipid layer or may be multilamellar. Suitableliposomes in accordance with the invention are preferably non-toxicliposomes such as, for example, those prepared from phosphatidyl-cholinephosphoglycerol, and cholesterol. The diameter of the liposomes usedpreferably ranges from 0.03-1.0 μm. However, other size ranges suitablefor phagocytosis by phagocytic cells may also be used.

[0062] In another embodiment, the therapeutic agent is embedded in acarrier (i.e., embedding agent) of desired properties. A therapeuticagent which is embedded includes those therapeutic agents that areembedded, enclosed, and/or adsorbed within a carrier, dispersed in thecarrier matrix, adsorbed or linked on the carrier surface, or acombination of any of these forms. In specific embodiments, theembedding agent (or carrier) is a microparticle, nanoparticle,nanosphere, microsphere, microcapsule, or nanocapsule (see e.g., M.Donbrow in: Microencapsulation and Nanoiparticles in Medicine andPharmacy, CRC Press, Boca Raton, Fla., 347, 1991). The term carrierincludes both polymeric and non-polymeric preparations. In a specificembodiment, the embedding agent is a nanoparticle. Preferably,nanoparticles are 0.03-1.0 microns in diameter and can be spherical,non-spherical, or polymeric particles. The therapeutic agent may beembedded in the nanoparticle, dispersed uniformly or non-uniformly inthe polymer matrix, adsorbed on the surface, or in combination of any ofthese forms. In a preferred embodiment, the polymer used for fabricatingnanoparticles is biocompatible and biodegradable, such aspoly(DL-lactide-co-glycolide) polymer (PLGA). However, additionalpolymers which may be used for fabricating the nanoparticles include,but are not limited to, PLA (polylactic acid), and their copolymers,polyanhydrides, polyalkyl-cyanoacrylates (such aspolyisobutylcyanoacrylate), polyethyleneglycols, polyethyleneoxides andtheir derivatives, chitosan, albumin, gelatin and the like.

[0063] In another embodiment, the therapeutic agent is in particulateform, the particles each being of desired properties. A particulatetherapeutic agent form includes any insoluble suspended or dispersedparticulate form of the therapeutic agent which is not encapsulated,entrapped or absorbed within a carrier. A therapeutic agent which is inparticulate form includes those therapeutic agents that are suspended ordispersed colloids, aggregates, flocculates, insoluble salts, insolublecomplexes, and polymeric chains of an agent. Such particulates areinsoluble in the fluid in which they are stored/administered (e.g.,saline or water) as well as the fluid in which they provide theirtherapeutic effect (e.g., blood or serum). Typically, “insoluble” refersto a solubility of one (1) part of a particulate therapeutic agent inmore than ten-thousand (10,000) parts of a solvent. Any method known inthe art to make particulates or aggregates can be used. Preferably,particulates are 0.03-1.0 microns in diameter and can be any particularshape.

5.2.1 Determination of Particle Size

[0064] Formulations containing therapeutic agents are preferablyprepared such that the size of the formulation is large enough to onlyor primarily be internalized by phagocytosis, that is, preferably largerthan 0.03 microns. In preferred embodiments, such formulations are0.03-1.0 microns, more preferably 0.07-0.5 microns, more preferably0.1-0.3 microns, and most preferably 0.1 to 0.18 microns. Any methodknown in the art can be used to determine the size of the formulationbefore administration to a patient in need thereof For example, a NicompSubmicron Particle Sizer (model 370, Nicomp, Santa Barbara, Calif.utilizing laser light scattering can be used.

5.3 Administration of the Formulation

[0065] Effective amounts of the formulations are contemplated as shortterm, acute therapy and are not meant for chronic administration. Timeperiod of treatment is preferably such that it producesinhibition/depletion of phagocytic cells for a period that is less thana month, preferably less than two weeks, most preferably up to one week.Empirically, one can determine this by administering the compound to anindividual in need thereof (or an animal model of such an individual)and monitoring the level of inhibition/depletion at different timepoints. One may also correlate the time of inhibition with theappropriate desired clinical effect, e.g. reduction in the acute risk ofplaque rupture.

5.4 Characterization of Therapeutic Utility

[0066] The term “effective amount” denotes an amount of a particularformulation which is effective in achieving the desired therapeuticresult, namely inhibited or decreased phagocytic cell activity and/orelimination or reduction in the amount of phagocytic cells. In oneembodiment, the desired therapeutic result of inhibiting or decreasingphagocytic cell activity and/or eliminating or reducing in the amount ofphagocytic cells stabilizes a vulnerable or unstable plaque in a patientsuffering from unstable angina. In another embodiment, the desiredtherapeutic result of inhibiting or decreasing phagocytic cell activityand/or eliminating or reducing in the amount of phagocytic cellsminimizes the infarct size and/or the amount of myocardial necrosis in apatient having suffered an acute myocardial infarction.

[0067] Toxicity and efficacy of the therapeutic methods of the instantinvention can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD₅₀(the dose lethal to 50% of the population), the No Observable AdverseEffect Level (NOAEL) and the ED₅₀ (the dose therapeutically effective in50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀ or NOAEL/ED₅₀. Formulations that exhibit large therapeuticindices are preferred. While formulations that exhibit toxic sideeffects may be used, care should be taken to design a delivery systemthat targets the agents of such formulations to the site of affectedtissue in order to minimize potential damage to unaffected cells and,thereby, reduce side effects.

[0068] The data obtained from the cell culture assays and animal studiescan be used in determining a range of dosage of the formulation for usein humans. The dosage of such formulations lies preferably within arange of circulating concentrations that include the ED₅₀ with little orno toxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anyformulation used in the method of the invention, the effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

[0069] The protocols and compositions of the invention are preferablytested in vitro, and then in vivo, for the desired therapeutic activity,prior to use in humans. One example, of such an in vitro assay is an invitro cell culture assay in phagocytic cells which are grown in culture,and exposed to or otherwise administered to cells, and observed for aneffect of this assay upon the cells, e.g., inhibited or decreasedactivity and/or complete or partial cell death. The phagocytic cells maybe obtained from an established cell line or recently isolated from anindividual as a primary cell line. Many assays standard in the art canbe used to measure the activity of the formulation on the phagocyticcells; for example, macrophage/monocyte activation can be assayed byquantitating the levels of chemotactic factors such as macrophagechemoattractant protein-1 (MCP-1), interleukin 1 beta (IL-1β), tissuenecrosis factor alpha (TNF-α) and macrophage inflammatory protein-1alpha (MIP-1 alpha). Many assays standard in the art can be used toassess survival and/or growth of the phagocytic cells; for example, cellproliferation can be assayed by measuring ³H-thymidine incorporation, bydirect cell count, by detecting changes in transcriptional activity ofknown genes such as proto-oncogenes (e.g., fos, myc) or cell cyclemarkers; cell viability can be assessed by trypan blue straining.

[0070] Selection of the preferred effective dose can be determined(e.g., via clinical trials) by a skilled artisan based upon theconsideration of several factors known to one of ordinary skill in theart. Such factors include the acute coronary syndrome to be managed ortreated, the symptoms involved, the patient's body mass, the patient'simmune status and other factors known to the skilled artisan to reflectthe accuracy of administered pharmaceutical compositions.

5.5 Pharmaceutical Compositions and Routes of Administration

[0071] Formulations comprising one or more therapeutic agents for use inthe methods of the invention may be in numerous forms, depending on thevarious factors specific for each patient (e.g., the severity and typeof disorder, age, body weight, response, and the past medical history ofthe patient), the number and type of therapeutic agents in theformulation, the type of formulation (e.g., encapsulated, embedded,particulate, etc.), the form of the composition (e.g., in liquid,semi-liquid or solid form), and/or the route of administration (e.g.,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, vaginal, orrectal means). Pharmaceutical carriers, vehicles, excipients, ordiluents may be included in the compositions of the invention including,but not limited to, water, saline solutions, buffered saline solutions,oils (e.g., petroleum, animal, vegetable or synthetic oils), starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, ethanol, dextrose and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like.

[0072] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances, which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. In addition, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyloleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0073] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, and the like. Saltstend to be more soluble in aqueous solvents, or other protonic solvents,than are the corresponding free base forms.

[0074] Pharmaceutical compositions can be administered systemically orlocally, e.g., near the site of pathology of an acute coronary syndrome.Additionally, systemic administration is meant to encompassadministration that can target to a particular area or tissue type ofinterest.

[0075] Pharmaceutical compositions are preferably administeredimmediately at the onset of the first symptoms of actual plaque rupture;such as, for example, chest pain, pain that radiates to the shoulder,arm, teeth, jaw, abdomen or back or shortness of breath or cough,lightheadedness, fainting, nausea, vomiting, sweating or anxietyassociated with a plaque rupture. Other symptoms will be apparent to theskilled artisan and medical doctor, and may be signals to administer theinstant pharmaceutical composition. Alternatively and/or additionally,the pharmaceutical compositions may be administered just after onset ofsymptoms, for example, within minutes of symptom onset. Alternativelyand/or additionally, the compositions may be administered within 1 hour,or about 2 hours, or about 3 hours or about 4 hours, or about 5 hours orabout 6 hours, up to within 1-3 days after onset of symptoms.

[0076] In another regime, pharmaceutical compositions are administeredto a patient with an increased risk of plaque rupture. For example, thecompositions of the invention may be administered to a patient prior toa procedure which increases the risk of plaque rupture, such as, forexample, an angioplasty procedure. It may be preferred to administer thecomposition up to 3 days before such a procedure. Also preferred,administration may be 1-6 hours before the procedure or within 1 hour ofthe procedure or less than 1 hour before or even within minutes of theprocedure. The skilled person can readily determine the appropriatetiming of administration depending on various physiological factors,specific to the individual patient, such as, for example, weight,medical history and genetic predisposition, as well as various factorswhich influence the anticipated risk of plaque rupture such ascomplexity of the procedure to be performed.

[0077] The contents of all published articles, books, reference manualsand abstracts cited herein, are hereby incorporated by reference intheir entirety to more fully describe the state of the art to which theinvention pertains.

[0078] As various changes can be made in the above-described subjectmatter without departing from the scope and spirit of the presentinvention, it is intended that all subject matter contained in the abovedescription, or defined in the appended claims, be interpreted asdescriptive and illustrative of the present invention. Modifications andvariations of the present invention are possible in light of the aboveteachings.

6. EXAMPLES

[0079] The following examples as set forth herein are meant toillustrate and exemplify the various aspects of carrying out the presentinvention and are not intended to limit the invention in any way.

[0080] 6.1 Effect of Liposomal Alendronate on the Size of the Zone ofInfarct

[0081] The effects of treatment with encapsulated bisphosphonates on thezone of infarct were studied in a rabbit AMI model. LiposomalAlendronate, approx. 0.150 μm in diameter was made using the followingoutline:

[0082] a. Dissolve lipids, DSPC, DSPG and cholesterol in 1/1ethanol/tert-butanol.

[0083] b. Dilute solvent into buffer containing Alendronate to generatelarge multilamellar vesicles (MLVs).

[0084] c. Extrude MLVs through 200 nm polycarbonate filters to generatelarge unilamellar 150±20 nm vesicles (LUVs).

[0085] d. Ultra-filtrate LUVs to remove un-encapsulated alendronate.

[0086] e. Sterile filter

[0087] Eight New Zealand White male rabbits, 2.5-3.5 kg B.W., were fednormal chow and water ad libitum. The rabbits were randomly administeredsaline (control) or liposomal alendronate (3 mg/kg, i.v.) as a singleinfusion simultaneous with coronary artery occlusion. The rabbits wereanesthetized by Ketamine/Xylazine (35 mg/kg; 5 mg/kg) and Isoflurane.The experiment was performed with respiratory support given byintubation and mechanical ventilation with isoflurane in balance oxygen,and continuous echocardiogram (ECG) and arterial blood pressure(catheter in ear artery) monitoring. Thoracotomy was performed throughthe left 4^(th) intercostal space, followed by pericardiotomy andcreation of a pericardial cradle. The left main coronary artery wasidentified and a large branch was encircled by a 5-0 silk suture and asnare. Thereafter, the snare was tightened for 30 minutes. Ischemia wasverified by ECG changes (ST-T segment elevation), changes of segmentcoloration and hypokinesia. After thirty minutes, the snare was releasedand resumption of blood flow was confirmed. The suture was left inplace, released, and the chest cavity was closed in layers. Buprenex wasadministered to the rabbits for analgesia for 2-3 additional days.Following euthanasia with Penthotal, the rabbits were sacrificed after 7days and the hearts were harvested. The coronary arteries were perfusedthrough the ascending aorta with saline, followed by tightening of thesuture on the previously occluded coronary artery and perfusion of thecoronary arteries with 0.5% Evans blue solution (Sigma) to stain areasof re-endothelialization (presence of blood). The left ventricular areaunstained by Evans blue was defined as the area at risk. The hearts werethen frozen at −20° C. for 24 hours and cut into transverse sections 2mm apart. Slices of the hearts were incubated for 30 minutes in thevital stain tritetrazolium chloride (TTC, 1%, Sigma), fixed in 10%natural buffered formalin to stain cells that had been alive previous totissue processing. The left ventricular area not stained by TTC (white)was defined as the area of infarct. The stained sections were thenphotographed and processed by digital planimetry (Photoshop).

[0088] Rabbits treated with liposomal alendronate had a zone of infarctthat was 29.5±6% of the area at risk. This was contrasted with thecontrol rabbits (untreated with liposomal alendronate) which showed aninfarct zone that was 42±5.5% of the area at risk (FIG. 1). Accordingly,liposomal alendronate was effective in reducing the zone of infarct. Noadverse effects were observed in the treatment group.

[0089] 6.2 Effect of Liposomal Alendronate On Myocardial Morphology

[0090] Rabbits as treated in Section 6.1 showed variation myocardialmorphology as exhibited by Hemotoxylin and Eosin staining. The controlrabbits have a distorted myocardial morphology (FIG. 2A) while therabbits treated with liposomal alendronate exhibit a more normalmorphology (FIG. 2B).

[0091] 6.3 Effect of Liposomal Alendronate on Macrophage Infiltration

[0092] Rabbits as treated in Section 6.1 showed a reduction inmacrophage infiltration in rabbits treated with liposomal alendronate.Representative sections of the rabbits' hearts were subjected toimmunostaining for RAM11+macrophages. Sections from rabbits treated withliposomal alendronate (FIG. 3B) showed less staining and therefore hadless RAM11+macrophages accumulation than sections from control rabbits(FIG. 3A).

[0093] Liposomal alendronate was also shown to reduce the number ofcirculating monocytes systemically. Rabbits were administered saline(control) or liposomal alendronate (3 mg/kg, i.v.) Monocyte levels incirculating blood were determined using FACS analysis for CD-14. At 48hours after injection with liposomal alendronate, the blood monocytepopulation was reduced by 75-95% as compared to the control group.

We claim:
 1. A method of treating an acute coronary syndrome comprisingadministering an effective amount of a formulation that decreasesphagocytic cell activity to a patient in need thereof, said formulationcomprising a therapeutic agent which is encapsulated, embedded, or in aparticle that is 0.05-1.0 microns in size.
 2. A method of treating anacute coronary syndrome comprising administering an effective amount ofa formulation that decreases the phagocytic cell numbers to a patient inneed thereof, said formulation comprising a therapeutic agent which isencapsulated, embedded, or in a particle that is 0.05-1.0 microns insize.
 3. The method of claim 1 or 2, wherein said acute coronarysyndrome is unstable angina.
 4. The method of claim 1 or 2, wherein saidacute coronary syndrome is impending or actual plaque rupture.
 5. Themethod of claim 1 or 2, wherein said acute coronary syndrome is acutemyocardial infarction.
 6. The method of claim 1 or 2, wherein saidtherapeutic agent is a bisphosphonate.
 7. The method of claim 6, whereinsaid bisphosphonate comprises a compound having formula (I):

wherein R₁ is H, OH or halogen group; and R₂ is halogen; linear orbranched C₁-C₁₀ alkyl or C₂-C₁₀ alkenyl, optionally substituted byheteroaryl or heterocyclyl C₁-C₁₀ alkylamino or C₃-C₈ cycloalkylamino,where the amino may be a primary, secondary or tertiary amine; —NHYwhere Y is hydrogen, C₃-C₈ cycloalkyl, aryl or heteroaryl; or —SZ, whereZ is chlorosubstituted phenyl or pyridinyl.
 8. The method of claim 6,wherein said bisphosphonate is selected from the group consisting ofclodronate, etidronate, tiludronate, pamidronate, alendronate andrisendronate.
 9. The method according to claim 1 or 2, wherein saidtherapeutic agent is encapsulated in a liposome.
 10. The method of claim1 or 2, wherein said therapeutic agent is embedded in a carrier selectedfrom the group consisting of microparticles, nanoparticles,microspheres, and nanospheres.
 11. The method of claim 1 or 2, whereinsaid therapeutic agent is formulated as a particulate selected from thegroup consisting of aggregates, flocculates, colloids, polymer chains,insoluble salts and insoluble complexes.
 12. The method of claim 1 or 2wherein more than one therapeutic agent is contained in the formulation.